N-furfurylphenylalanine

ABSTRACT

1. N-FURFURYLPHENYLALANINE.

United States Patent 2 Claims ABSTRACT OF THE DISCLOSURE The followingN-substituted amino acids were found to combat infection with influenzavirus in vivo:

N-ot-naphthylacetylleucine N-4-nitrophenoxyacetyl-4-nitr0phenylalanineN-carboxymethylphenylalanine N-benzylphenylalanineN-furfurylphenylalanine N-fl-naphthylaminomethylalanine N-finaphthylaminomethylmethionine 6- ,B-naphthylaminomethyl -aminocaproicacid N-lauroylphenylalanine N-4-toluenesulfonyltryptophanN-2-fluorenesulfonyl-fi-alanine N-Z-fiuorenesulfonylvalineN-2fluorenesulfonyl-4-nitrophenylalanine This application is a divisionof the copending application Ser. No. 69,993, filed Sept. 4, 1970, andnow Pat. No. 3,801,633.

This invention relates to certain N-substituted amino acids and to theiruse as therapeutic agents in combating infection with influenza virus.

It has been found that N-u-naphthylacetylleucine, N-4 nitrophenoxyacetyl4 nitrophenylalanine, N-carboxymethylphenylalanine,N-benzylphenylalanine, N-furfurylphenylalanine, N,8-naphthylaminomethylalanine, N-B- naphthylaminomethylmethonine, 6 (BnaphthylaminomethyD-aminocaproic acid, N-lauroylphenylalanine, N-4-toluenesulfonyltryptophan, N-2 fluorenesulfonyl t3 alanine, N 2fluorenesulfonylvaline, and N-Z-fluorenesulfonyl-4-nitrophenylalanineare effective in viva against influenza virus in amounts in which theirtoxicity is low. As will be shown in more detail hereinafter, thecompounds of the invention compare favorably in their therapeutic indexvalues with the known anti-viral compounds Amantadine and Rimantadine.

The compounds of the invention may be employed in the free acid form orin the form of their pharmaceutically acceptable salts, particularly thealkali metal salts, the alkaline earth metals salts, organic amine saltsand the like, such as the sodium, potassium, magnesium, calcium,ammonium, monoethanolamine and diethanolamine salts which are generallymore soluble in water than the free acids, and are not more toxic thanthe free acids in equimolecular amounts.

The compounds of the invention are administered orally or parenterallyin therapeutic compositions which contain one or more of the compoundsas active agents in combination with a pharmaceutical carrier which maybe solid or liquid, depending on the intended mode of application.Liquid compositions may be formulated in a conventional manner for oralapplication or for injection in dosage unit form.

The solid compositions, such as tablets, pills, powders, or granules,are prepared by combining the active agents of the invention with solidpharmaceutical carriers conventional in themselves, such as calciumcarbonate, lac- 3,850,968 Patented Nov. 26, 1974 tose, sucrose,sorbitol, mannitol, starch, amylopectin, methylcellulose, or gelatin.Compacting of the comminuted ingredients may be facilitated by the useof mag nesium stearate, calcium stearate, or polyethylene glycol.

Orally applicable liquid compositions may be emulsions, solutions, orsuspensions of the active agents in water, liquid paraifin and likepharmaceutically acceptable inert liquids which may additionally containsurface active agents to keep the anti-viral agents and otheringredients dispersed in the liquid carrier, and adjuvants for improvingthe taste or odor of the composition, as is well known in itself.

In preparing compositions of the invention for parenteral application,it is preferred. to disperse the N-substituted amino acids of theinvention in sterilized water containing enough alkaline material todissolve the active agents, and to transfer dosage units of the solutionto vials which are sealed thereafter.

The compounds of the invention differ from each other in theirtherapeutic effects and in their toxicity, and the desired therapeuticeffects may be achieved With some by a dosage of 200 mg. per day appliedorally or 400 mg. per day applied parenterally, whereas others may needto be administered orally at a rate of 1,000 mg. per day or parenterallyat 2,000 mg. per day. A single daily dose is preferred.

Those compounds of the invention which exist either in optically active(D or L) forms or in the optically inactive racemic form are equallyeffective in all forms.

Only N-carboxymethylphenylalanine, N-benzylphenylalanine,N-lauroylphenylalanine, and N-4-toluenesu1fonyltryptophan were knownheretofore. The other nine N- substituted amino acids enumerated aboveare new, but are readily prepared by means of procedures known inthemselves. The Schotten-Baumann reaction may be used in preparing theN-a-naphthylacetyl derivative of leucine, the N 4-nitrophenoxyacetylderivative of 4-nitrophenylalanine, and the N 2-fluorenesulfonylderivatives of ,8- alanine, valine, and 4-nitropheny'lalanine. Furfuralmay be reacted with phenylalanine in an aqueous medium in the presenceof sodium borohydride to N-furfurylphenylalanine, and fi-naphtylaminemay be reacted with alanine, methionine, or 6-aminocaproic acid in thepresence of formaldehyde in preparing the N-B-naphthylaminomethylderivatives of the last-mentioned amino acids.

The following Examples further illustrate the preparation of theN-substituted amino acids of this invention, of their salts, and ofcompositions containing the active agents of the invention.

EXAMPLE 1 A mixture of 9.3 g. a-naphthaleneacetic acid and 4.6 g.phosphorus trichloride was heated for about one hour on a water bathmaintained at C. The mixture was then cooled, the supernatant liquid wasdecanted from the precipitated a-naphtylacetyl chloride, and the latterwas dissolved in 30 ml. anhydrous ether.

The ether solution was added dropwise over a period of 20 minutes withstirring to a solution of 6.3 g. L-leucine in 20 ml. 10% sodiumhydroxide solution mixed with a solution of 15.9 g. sodium carbonate inml. water at room temperature. Stirring was continued for three hours,and the reaction mixture was then made acid to Congo Red with 6 Nhydrochloric acid. The precipitated crystals ofN-u-naphthylacetyl-L-leucine were filtered out, washed with water, andrecrystallized from aqueous ethanol. 11.8 g. Columnar crystals (78%yield) melting at 152 C. were obtained. [a] =22.O7 (c.=1.675, absoluteethanol).

The compound was identified by elementary analysis. Calculated for C H ON 72.21% C; 7.07% H; 4.67% N. Found: 72.29% C; 7.16% H; 4.74% N.

3 EXAMPLE 2 A mixture of 4 g. 4-nitrophenoxyacetic acid and 20 ml.thionyl chloride was refluxed for one hour, and the unreacted thionylchloride was removed by vacuum distillation. The residue of4-nitrophenoxyacetyl chloride was dissolved in anhydrous ether.

Over a period of 20 minutes, the ether solution was added dropwise andwith vigorous stirring to a solution of 4.2 g. DL-4-nitrophenylalaninein 20 ml. sodium hydroxide mixed with a solution of 5.3 g. sodiumcarbonate in 50 ml. water. Stirring was continued after the addition forthree hours, and crystalline N-4-nitrophen0xyacetyl-DL-4-nitrophenylalanine was precipitated by acidifying as in Example 1.After recrystallizing from aqueous dioxane, 6.0 g. of needle-shapedcrystals were obtained (77% yield) and melted at 184 C. The compound wasidentified by elementary analysis.

Calculated for C17H150 N3: 10.79% N Found: 52.69 C; 3.99% H; 10.63% N.

EXAMPLE 3 A mixture of 16.5 g. L-phenylalanine, 8 g. sodium hydroxide,100 ml. water, and 9.5 g. monochloroacetic acid was heated on a steambath for two hours. N-Carboxymethyl-L-phenylalanine was precipitatedfrom the reaction mixture with 38% hydrochloric acid, filtered off,washed with water and ether, and recrystallized from water. Flat needlesmelting at 243 C. were obtained in a yield of 17.4 g. (90%). [a]=+15.6'/ (c.=1.85, 1N HCl).

Elementary analysis: Calculated for C11H1304N: N. Found: 59.18% C; 5.92%H; 6.04% N.

EXAMPLE 4 As in Example 3, 16.5 g. L-phenylalanine, 6 g. sodiumhydroxide, 50 ml. water, 45 ml. ethanol, and 13 g. benzyl chloride werekept on a water bath at 100 C. for four 4 weighed 22.5 g. (46% yield)and melted at 241 C.

hours, and the precipitated and recovered N-benzyl-L- phenylalanine waswashed with water, ether, and ethanol and recrystallized from 40% aceticacid. The purified, needle-shaped crystals weighed 10.7 g. (42% yield)and melted at 234 C. [a] =i+18.09 (c.'=1.006, 0.2 N NaOH).

Elementary analysis: Calculated for C16H1702N: 75.25% C; 6.71% H; 5.49%

N. Found: 75.34% C; 6.61% H; 5.58% N.

EXAMPLE 6 Elementary analysis: Calculated for C H NO 68.53% C; 6.16% H;5.71%

N. Found: 68.19% C; 6.15% H; 5.66% N.

EXAMPLE 7 A solution of 4.29 g. fl-naphthylamine in 35 ml. 60% ethanolwas added to a mixture of 2.67 g. L-alanine, 9 ml. 37% formaldehydesolution, and 35 ml. 60% ethanol. A small amount of amorphousprecipitate was removed by filtering, and the filtrate was evaporated ina vacuum. The residue was mixed with acetone and stored in arefrigerator to precipitate crystals ofN-[i-naphthylaminomethyl-L-analine which were filtered 01f, washed withacetone, and recrystallized from dimethylsulfoxide. The purifiedcolumnar crystals weighed 2.45 g. (50% yield) and melted at 264 C. [a]=25.92 (c.=0.54, 0.2 N NaOH).

Elementary analysis: Calculated for C H O N 68.81% C; 6.60% H; 11.47%

N. Found: 70.84% C; 5.72% H; 11.03% N.

EXAMPLE 8 14.9 g. DL-Methionine, 30 ml. 37% formaldehyde solution, and150 ml. 33% ethanol were kept at 50 C. until a homogeneous solution wasformed to which a solution of 14.3 g. ,B-naphthylamine in ml. 60%ethanol was added. The resulting reaction was exothermic and a smallamount of amorphous material precipitated and was filtered oil. Thefiltrate was stored for several days, whereby crystals ofN-fl-naphthylaminomethyl-DL- methionine were precipitated. They wererecovered by filtration, washed with 50% ethanol, and dissolved inaqueous ammonia. A small amount of insoluble material was removed byfiltering, and the filtrate was barely acidified with dilute acetic acidto reprecipitate purified, platelet-shaped crystals which were filteredoff, washed with water and acetone, and dried. They weighed 3.8 g. (66%yield) and melted at 235 C.

Elementary analysis: Calculated for C H O N S: 63.13% C; 6.58% H;

9.21% N. Found: 63.79% C; 6.24% H; 8.79% N.

EXAMPLE 9 A solution of 14.3 g. fi-naphthylamine in 100 ml. 60% ethanolwas added to 13.1 g. 6-arninocaproic acid and 30 ml. 37% formaldehydesolution in 125 ml. 60% ethanol. An exothermic reaction occurred and asmall amount of insoluble material was filtered off. The filtrate wasevaporated in a vacuum until crystals precipitated. The precipitate wasrecovered by filtering, washed with acetone, and recrystallized fromabsolute ethanol. The purified, plate-shaped crystals of6-(fl-naphthylaminomethyl)aminocaproic acid weighed 20.3 g. (81% yield)and melted at 127 C.

Elementary analysis: Calculated for C H O N 69.72% C; 7.02% H; 10.80% N.Found: 70.44% C; 7.03% H; 10.41% N.

EXAMPLE 10 22 g. Lauroyl chloride in 44 ml. ether were added at roomtemperature to a solution of 16.5 g. L-phenylalanine and 16 g. sodiumhydroxide in ml. water.

v The mixture was stirred for three hours and then acidified to CongoRed with dilute hydrochloric acid. The precipitated crystals ofN-lauroyl-L-phenylalanine were filtered 01f, washed with water, andrecrystallized from acetone. They were then plate-shaped, weighed 25 g.(73% yield), and melted at 97 C. [a] =+20.00 (c.=l.05, absoluteethanol).

Elementary analysis:

Calculated for C H O N: 72.56% C; 9.57% H; 4.03%

N. Found: 71.71% C; 9.63% H; 3.93% N.

5 EXAMPLE 11 A solution of 9.5 g. 4-toluenesulfonyl chloride in 50 ml.ether was added dropwise to a solution of 102g. DL-tryptophan and 12 g.sodium hydroxide in 120 ml. water over a period of 40 minutes withstirring. The reaction mixture was stirred for three hours more andacidified with hydrochloric acid to precipitateN-4-toluenesulfonyl-DL-tryptophan. The crystals recovered by filteringwere washed with water and recrystallized from ethanol. They weighed10.5 g. (59% yield), were needleshaped, and melted at 184 C.

Elementary analysis:

Calculated for C H O N 60.31% C; 5.06% H;

7.81% N. Found: 60.47% C; 5.19% H; 7.67% N.

EXAMPLE 12 A solution of 5.3 g. 2-fiuorenesulfonyl chloride in 150 ml.acetone was added dropwise over a period of 30 minutes with stirring toa solution of 1.8 g. fi-alanine in a mixture of 10 ml. 10% sodiumhydroxide and a solution of 5 .3 g. sodium carbonate in 50 ml. water atroom. temperature. Stirring was continued for 30 minutes on a water bathat 50 C., and for three hours more at room temperature. When thereaction mixture was then acidified with dilute hydrochloric acid andacetone removed by vacuum distillation, N-2-fiuorenesulfonyl-fi-alaninecrystallized. The crystals were filtered off, washed with water, andrecrystallized from dioxane. The purified, plate-shaped crystals weighed5.5 g. (87% yield) and melted at 187 C.

Elementary analysis:

Calculated for C H O NS: 60.55% C; 4.76% H;

4.41% N. Found: 60.54% C; 4.58% H; 4.30% N.

EXAMPLE 13 A solution of 8 g. 2-fluorenesulfonyl chloride in 240 ml.acetone was added dropwise with stirring at room temperature to asolution of 3.5 g. L-valine in a mixture of 20 ml. 10% sodium hydroxidesolution and 5.3 g. sodium carbonate in 50 ml. water. The reactionmixture was stirred at 50 C. for 30 minutes on a water bath, and forthree additional hours at room temperature, whereupon it was acidifiedand partly evaporated in a vacuum to remove the acetone and to inducecrystallization of N-2-fluorenesulfonyl-L-valine. The crystals werefiltered 01f, washed with water, and recrystallized from dilute acetone.The purified columnar crystals weighed 7.0 g. (70% yield) and melted at220 C. [u] =+22.33 (c.=2.060, acetone).

Elementary analysis:

Calculated for C H O NS: 62.58% C; 5.54% H; 4.05% Found: 62.43% C; 5.57%H; 3.77% N.

EXAMPLE 14 A solution of 5.3 g. 2-fluorenesulfonyl chloride in 150 ml.acetone was stirred dropwise over a period of 30 min utes into asolution of 4.2 g. DL-4-nitrophenylalanine in a mixture of 20 ml. 10%sodium hydroxide solution and 5.3 g. sodium carbonate in 50ml. water atroom tempera ture. The reaction mixture was thereafter stirred fr,30minutes at 0 C. on a water bath and then for three hours at roomtemperature. Upon acidifying with dilute hydrochloric acid andevaporation of the acetone, crystallineN-2-fluorenesulfonyl-DL-4-nitrophenylalanine was precipitated. Thecrystals were filtered off, washed with water,

and recrystallized from dioxane. The purified, very small.

crystals weighed 5 g. (57% yield) and melted at 261 C.

Elementary analysis: Calculated for C H O N S: 60.21% C; 4.13% H;

6.38% N. Found: 60.06% C; 4.31% H; 6.12% N.

6 'EXAMPLE 15 2.33 g. (0.0 1 mole) N-carboxymethyl-L-phenylalanine wasadded to a 5% aqueous solution containing 0.84 g. (0.01 mole) sodiumbicarbonate to form a clear solution. The solution was concentratedunder reduced pressure to crystallize out sodium salt ofN-carboxymethyl-L-phenylalanine.

The sodium salts of the other thirteen N-substituted amino acids wereprepared by the same manner.

EXAMPLE 16 2.55 g. (0.01 mole) N-benzyl-L-phenylalanine was added to a10% aqueous solution containing 0.56 g. potassium hydroxide to form aclear solution. And then the solution was concentrated under reducedpressure to crystallize out potassium salt of N-benzyl-L-phenylalanine.

The potassium salts of the other thirteen N-substituted amino acids wereprepared by the same manner.

EXAMPLE 17 2.45 g. (0.01 mole) N-furfuryl-L-phenylalanine was added toan excess amount of 28% concentrated aqueous ammonia solution to form aclear solution. The solution was concentrated under reduced pressure tocrystallize out ammonium salt of N-furfuryl-L-phenylalanine.

The ammonium salts of the other thirteen N-substituted amino acids wereprepared by the same manner.

EXAMPLE 18 One thousand capsules for oral use, each containing 200 mg.of Compound 3 (N-carboxymethyl-L-phenylalanine as prepared in Example 3)were prepared from the following types and amounts of materials:

G. Compound 3 200 Corn starch 150 Talc Magnesium stearate 2.6

The materials were thoroughly mixed and then capsulated in the usualmanner. The foregoing capsules are useful for the treatment of infectionwith influenza virus by the oral administration of 1 to 2 capsules every6 hours.

This procedure may be similarly applied to prepare capsules containingone or more: of the other thirteen N-substituted amino acids.

EXAMPLE 20 One thousand tablets for oral use, each containing 200 mg. ofCompound 3 were prepared from the following type and amounts ofmaterials:

G. Compound 3 200 Lactose 50 Cornstarch 30 Magnesium stearate 3.0 Lightliquid petrolatum 1.2

The ingredients were thoroughly mixed and slugged. The slugs were brokendown by forcing through a number sixteen screen. The resulting granuleswere then compressed into tablets, each tablet containing 200- mg.Compound 3.

The above procedure may be similarly applied to prepare tabletscontaining one or more of the other thirteen N-substituted amino acids.

EXAMPLE 21 A sterile aqueous solution for intramuscular use, containingin 1 ml. 200 mg. of Compound 3, as the sodium salt was prepared from thefollowing types and amount of ingredients:

Compound 3 mg 200 Sodium hydroxide percent solution Water for injectionml 1000 Compound 3 was added to the water and sufficient sodiumhydroxide was added to form a solution of pH 7.2. The solution wassterilized by filtration. The sterile solution in the amount of 1 ml.was aseptically filled into sterile vials and frozen. The water wasremoved under high vacuum and the vials containing the lyophilizedpowder were sealed. Just prior to use, sufficient sterile water forinjection to make 1 ml. of solution was added to the vial.

The above procedure may be similarly applied to prepared parenteralsolution containing sodium salt of the other N-substituted amino acid.

8 EXAMPLE 23 5Mouse died within 5 days after infection and entire lungwas consolidated.

4Mouse survived at least 5 days, but entire lung was consolidated.

375% of lung consolidated.

250% of lung consolidated.

125% of lung consolidated.

O.55-15% of lung consolidated.

Table 2 lists the mean values and standard error of scores for eachgroup of ten mice and the dosage employed which was approximately /5 ofLD TABLE 2 Compound- Aman- Con- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 tadinetrol Dosage, mg./kg 60 100 300 300 300 200 300 300 200 170 80 80 300 60Mean Score..-" 2. 6 2. 6 2. 5 2. 2 2. 6 2. 2 2. 6 2. 8 2. 5 2. 0 2. 6 2.8 2. 6 2. 0 2. 6 4. 6 Standard error. 1. 7 1. 7 1. 7 1. 1 1.8 1. 31.4 1. 2 1. 6 1.4 1. 2 1. 2 1. 0 1. 3 2. 0 1. 9

These compositions and analogous compositions containing the otheractive agents of the invention were used successfully in the treatmentof many influenza strains, the results being consistent with thosespecifically described hereinafter with reference to two particularlyvirulent strains.

The physiological effects of the compounds of the invention areillustrated by the following Examples:

EXAMPLE 22 The several N-substituted amino acids of the inventionprepared as described in Examples 1 to 14 were dissolved in diluteaqueous sodium bicarbonate, the solutions were sterilized by passagethrough a microporous filter, and single doses of varying strength wereinjected intraperi toneally into mice in a standard toxicity test, malemice weighing 10l2 g. each being employed in all tests described below.The mean lethal dosage (LD was determined one week after the injection.The results obtained are shown in Table 1 in which the severalN-substituted amino acids of the invention are identified by numbersreferring to Examples 1-14 in which their preparation is described. Forcomparison purposes, the known antiviral agent Amantadine(l-Adamantanamine) was also tested.

TABLE 1 Compound: LD' Q, mg./kg. 1 300 13 375 14 1500 Amandtadine 233EXAMPLE 24 In an additional test series, groups of ten mice infected asin Example 23 were treated 72 hours after the infection byintraperitoneal injection with the compounds of the invention andAmantadine in the dosages listed in Table 2. Ten hours later, theinjections were repeated. A control group of mice received physiologicalsaline solution only, and an additional comparison group received 6Omg./kg. of Rimantadiene, another known antiviral agent.

The mice were sacrificed 96 hours after the last injection. The lungswere removed, and their virus content was estimated by standarddetermination of the mean Egg Infective Dosis (EID The values of EIDwere below 10 for each of the fourteen tested compounds of theinvention, 10 for Rimantadine, and 10 for Amantadiene as well as for theuntreated control group.

The results of Examples 23 and 24 show that the antiviral effects of thecompounds of the invention in doses of approximately equal toxicitycompare favorably with known antiviral agents when applied to standardlaboratory mice if the treatment is given 48 hours after the infection,but that the compounds of the invention are still effective when applied72 hours after infection whereas the known agents have only minimal orunmeasurable effects under such conditions.

EXAMPLE 25 The antiviral effects of the 14 compounds of the inventiontoward influenza B type Lee strain virus were also tested under thesimilar condition as in Example 23.

Groups of ten mice each were inejected intraperitoneally with singledoses of the 14 compounds of the invention, 48 hours after having beeninfected intranasally with influenza B type Lee strain virus. The micesurviving 14 days after the infection were sacrificed, and the lungs ofall tested mice were inspected for consolidation. The observations madeevaluated according to Ledinkos criteria described already. The dosageemployed was approximately /5 of LD The results were as shown in Table3.

TABLE 3 Compound- Aman- Con- 2 3 4 5 6 7 8 9 10 11 12 13 14 tadine trolDosage, mg./kg 100 300 300 300 200 300 300 200 170 80 300 60 Mean score.2. 5 2.1 2. 7 2.1 1. 8 2.0 2. 8 2. 2 2. 4 2. 0 2.1 2. 0 1. 9 4.1 4. 2Standard error. 1. 4 1. 3 1. 5 1. 2 1. 4 1.0 1. 8 1. 8 1. 4. 1. 2 1.0 1.3 1. 2 1.8 1. 8

The results show that the known antiviral agent exhibits no effecttoward influenza virus of B types whereas the compounds of the inventionexhibit antiviral effects 10 pounds whose LD values are higher than 1500mg./kg.

while it was approximately /5 of LD for the other compounds.

TABLE 4 Compound- Arnan- Con- 1 2 3 4 5 6 7 s 9 10 11 12 13 14 tadinetrol D0sage,mg./kg 300 300 300 300 300 300 300 300 300 250 250 300Meanseorenn 3.0 2.9 3.2 3.6 3.2 3.0 2.0 3.4 3.8 3.2 3.0 3.1 2.9 3.4 4.8Standarderror 1.2 1.0 1.2 1.4. 1.2 1.2 1.3 1.0 1.2 1.1 1.4 1.3 1.2 1.31.3

approximately equal with those toward influenza virus What is claimedis: of A-2 type. 1. N-furfurylphenylalanine.

EXAMPLE 26 25 2. N-furfuryl-L-phenylalanine.

Each of the 14 compounds of the invention was suspended incarboxyrnethylcellulose and the suspension was orally administered intothe ten mice infected as in Example 23 using a metallic tube, 48 hoursafter the infection. The mice surviving 14 days after the infection weresacrificed and the lungs of all tested mice were inspected forconsolidation. The results were as shown in Table 4. The dosage employedwas 300 mg./kg. for the com US. Cl. X.R. 424--285

1. N-FURFURYLPHENYLALANINE.